The effects of ascorbic acid on the intracellular metabolism of iron and ferritin

An important property of ascorbic acid is its ability to increase the availability of storage iron to chelators. To examine the mechanism of this effect, K562 cells were incubated with ascorbate, attaining an intracellular level of 1 nmol/10(7) cells. In contrast to the reductive mobilization of iron seen with isolated ferritin, ascorbate stabilized iron preincorporated into cellular ferritin. Biosynthetic labeling with [35S]methionine demonstrated that ascorbate also retarded the degradation of the ferritin protein shell. Ferritin is normally degraded in lysosomes. The lysosomal protease inhibitors leupeptin and chloroquine produced a qualitatively similar stabilization of ferritin. Ascorbate did not act as a general inhibitor of proteolysis, however, since it did not effect hemoglobin degradation in these cells. The stabilization of cellular ferritin by ascorbate was accompanied by an expansion of the pool of chelatable iron.     

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.     

Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.     

A simple assay for determination of iron release from ferritin in neuroblastoma cells.

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.     

Analysis of iron-containing compounds in different compartments of the rat liver after iron loading

Summary The livers of iron-loaded rats were fractionated and a cytosolic fraction, a lysosomal fraction, a siderosomal fraction and haemosiderin were obtained. All iron-containing compounds from these fractions were isolated and their morphology, Fe/P ratios, iron core diameter and peptide content were compared. The cytosolic fraction contained ferritin (CF) and a slower sedimenting, light ferritin (CLF). The lysosomal fraction also contained ferritin (LF) and a slower sedimenting light ferritin (LLF). The siderosomal fraction contained ferritin (SF), a faster sedimenting non-ferritin iron compound (SIC) and haemosiderin (HS). SIC and HS did not resemble ferritin as much as the other products did, but were found to be water-insoluble aggregates. The Fe/P ratios of CF and CLF were lower than the Fe/P ratios of LF and LLF and these in turn had lower Fe/P ratios than SF, SIC and HS. The iron core diameter of the cytosolic ferritin was increased after lysosomal uptake. The iron core diameters of the siderosomal products were smaller. CLF, CF, LF, LLF and SF contained one kind of subunit of approximately 20.5 kDa. SIC and HS contained other peptides in addition to the 20.5-kDa subunit. The results indicate that storage of ferritin molecules is not limited to the cytosolic compartment, but is also the case in the lysosomes. Extensive degradation of the ferritin molecule seems to be confined to the siderosomes.     

Transport of siderotic Kupffer cells to the lung and bronchial excretion of iron in experimental iron-overload.

In 3,5,5-trimethylhexanoylferrocene-induced iron overload of rats, three different types of iron-loaded macrophages and derivatives thereof were found in the lungs. On the basis of their localization and of their pattern of iron load it was possible to distinguish: (1) Resident macrophages, showing an alveolar localization and a moderate iron content represented by lysosomal ferritin and haemosiderin. (2) Liver-derived macrophages and giant cells, as well as fragments of them. They showed an exclusive localization in capillaries and alveolar septa, and high concentrations of free ferritin molecules in addition to polymorphous ferritin- and haemosiderin-containing siderosomes. (3) Monocyte-derived intravascular pulmonary macrophages. Initially, they contained iron only as lysosomal aggregates of ferritin and haemosiderin, as a result of phagocytosis of liver-derived macrophageal cell fragments. Later in iron overload, they also showed free ferritin molecules in the cytosol and fused intrapulmonarily to giant cells. The resident as well as the liver-derived siderotic pulmonary macrophages provide a way for iron excretion through the airways.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Effect of ferritin-containing fractions with different iron loading on lipid peroxidation.

Ferritin-containing fractions with different degrees of iron loading were prepared. All ferritin fractions stimulated the peroxidation of bovine brain phospholipid liposomes, as measured by the formation of thiobarbituric acid-reactive material. This stimulation was increased in the presence of ascorbate. Iron salts of equivalent concentration to those of the ferritin fractions were more stimulatory to lipid peroxidation at the higher iron concentrations. None of the fractions inhibited ascorbate-dependent peroxidation in the presence of added iron salts.     

Overexpression of transferrin receptor and ferritin related to clinical symptoms and destabilization of human carotid plaques

Accumulation of tissue iron has been implicated in development of atherosclerotic lesions mainly because of increased iron-catalyzed oxidative injury. However, it remains unknown whether cellular iron import and storage in human atheroma are related to human atheroma development. We found that transferrin receptor 1 (TfR1), a major iron importer, is highly expressed in foamy macrophages and some smooth muscle cells in intimal lesions of human carotid atheroma, mainly in cytoplasmic accumulation patterns. In 52 human carotid atherosclerotic lesions, TfR1 expression was positively correlated with macrophage infiltration, ectopic lysosomal cathepsin L, and ferritin expression. Highly expressed TfR1 and ferritin in CD68-positive macrophages were significantly associated with development and severity of human carotid plaques, smoking, and patient's symptoms. The findings suggest that pathologic macrophage iron metabolism may contribute to vulnerability of human atheroma, established risk factors, and their clinical symptoms. The cytoplasmic overexpression of TfR1 may be the result of lysosomal dysfunction and ectopic accumulation of lysosomal cathepsin L caused by atheroma-relevant lipids in atherogenesis.     

Erythropoietin effects on iron metabolism in rat bone marrow cells

This paper describes a study of the incorporation of ^{5 9}Fe from ^{5 9}Fe-labelled rat transferrin into rat bone marrow cells in culture. ^{5 9}Fe was found in both stroma and cytoplasm of marrow cells, and the cytoplasmic ^{5 9}Fe separated by polyacrylamide gel electrophoresis, into ferritin, haemoglobin and a low molecular weight fraction.The incorporation of ^{5 9}Fe into all three cytoplasmic fractions, but not into the stroma, increased progressively with time. Erythropoietin stimulated the increase of ^{5 9}Fe in ferritin within 1 h, the earliest time examined, and more than 3 h later in the stroma and haemoglobin.A proportion of the ⁵⁹Fe incorporated into the stroma and low molecular weight iron fractions during a 1 h incubation with ⁵⁹Fe-labelled transferrin was mobilised into ferritin and haemoglobin during a subsequent 4-h “cold-chase”. Erythropoietin, when present during the “cold-chase”, did not influence these ⁵⁹Fe fluxes. The erythropoietin stimulation of ⁵⁹Fe incorporation into ferritin, one of the earliest erythropoietin effects to be recorded, was therefore considered to be due to an increase of ⁵⁹Fe uptake by the hormone-responsive cells rather than a direct effect on ferritin synthesis.20-h cultures containing erythropoietin when incubated with ⁵⁹Fe-labelled transferrin for 4 h, showed dose-related erythropoietin stimulation of ⁵⁹Fe incorporation into haemoglobin only.In the presence of 10 mM isonicotinic acid hydrazide, ⁵⁹Fe incorporation into haemoglobin was inhibited, as in reticulocytes (Ponka, P. and Neuwirt, J. (1969) Blood 33, 690–707), while that into the stroma, ferritin and low molecular weight iron fractions, was stimulated; there were no reproducible effects of erythropoietin.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption!

Ascorbate is a cofactor in numerous metabolic reactions. Humans cannot synthesize ascorbate owing to inactivation of the gene encoding the enzyme l-gulono-γ-lactone oxidase, which is essential for ascorbate synthesis. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance nonheme iron absorption in the gut, ascorbate within mammalian systems can regulate cellular iron uptake and metabolism. Ascorbate modulates iron metabolism by stimulating ferritin synthesis, inhibiting lysosomal ferritin degradation, and decreasing cellular iron efflux. Furthermore, ascorbate cycling across the plasma membrane is responsible for ascorbate-stimulated iron uptake from low-molecular-weight iron–citrate complexes, which are prominent in the plasma of individuals with iron-overload disorders. Importantly, this iron-uptake pathway is of particular relevance to astrocyte brain iron metabolism and tissue iron loading in disorders such as hereditary hemochromatosis and β-thalassemia. Recent evidence also indicates that ascorbate is a novel modulator of the classical transferrin–iron uptake pathway, which provides almost all iron for cellular demands and erythropoiesis under physiological conditions. Ascorbate acts to stimulate transferrin-dependent iron uptake by an intracellular reductive mechanism, strongly suggesting that it may act to stimulate iron mobilization from the endosome. The ability of ascorbate to regulate transferrin iron uptake could help explain the metabolic defect that contributes to ascorbate-deficiency-induced anemia.     

Intracellular transport and degradation of I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Alteration of lysosomal density by sequestered glycogen during deprivation-induced autophagy in rat liver.

Livers from fed rats were perfused in the single-pass mode with and without 10 mM glucose; autophagy then was induced by deleting amino acids. The decrease in glycogen which occurred in the absence of glucose did not influence the magnitude of the autophagic response, but it did affect the composition of autophagic vacuoles and the distribution of lysosomal marker on isopycnic centrifugation. In livers undepleted of glycogen, amino acid omission shifted a substantial portion of the β-acetylglucosaminidase peak into heavier gradient fractions. This shift was reduced 50% in partially depleted livers and was accompanied by a 40% decrease in glycogen-containing particles. These findings support the notion that glycogen sequestered during autophagy is responsible for the enhanced lysosomal density.     

Quantitative determination of intracellular, ferritin-associated radioactive iron by high-performance liquid chromatography and immunoprecipitation

Antibodies raised against ferritin preparations of diverse origin provide an uncertain reagent for quantitation of the ferritin present in specific cell lysates. Utilizing K562 cells, a human leukemic cell line, techniques are described to resolve and to quantitate the ferritin-bound cytosolic iron. Processing the cell lysates by HPLC employing an anion-exchange or hydrophobic interaction column resulted in recovery of a single, ferritin-containing radioactive peak widely separated from the bulk of the non-ferritin-bound iron. Comparison of the yield obtained by chromatography with that by immunoprecipitation confirmed both the specificity and the quantitation of the antibody technique.     

INTRANUCLEAR AGGREGATES OF FERRITIN IN LIVER CELLS OF MICE TREATED WITH SACCHARATED IRON OXIDE : Their Possible Relation to Nuclear Protein Synthesis

Several months following parenteral injections of saccharated iron oxide into DBA/2J mice, granules rich in iron were found in nuclei of scattered parenchymal liver cells as well as in the cytoplasm. As seen in the light microscope, the intranuclear granules were brown; most of them measured between 0.5 µ and 1 µ in cross-section. They gave positive Prussian blue tests, and were not selectively stainable with pyronine. Electron micrographs of the granules showed closely packed aggregates of ferritin molecules, occasionally in paracrystalline order. The intranuclear collections were often surrounded by bands of material of moderate opacity. Scattered ferritin molecules and collections of such molecules were also present in the cytoplasm of many liver cells, but there seemed to be no quantitative relationship between intranuclear and cytoplasmic ferritin. Liver cells from untreated control mice failed to reveal intranuclear deposits of ferritin. Although the site of origin of the intranuclear aggregates of ferritin is unknown, the findings suggest the possibility that under suitable circumstances ferritin synthesis may take place within nuclei of liver cells—perhaps induced by the presence of colloidal iron.     

Origin and composition of settling iron aggregates in oligotrophic Sombre Lake, Signy Island, Antarctica

Sediment traps were deployed in an oligotrophic, seasonally anoxic maritime Antarctic lake for 15 months. Immediately after the onset of the inflow in spring many iron oxyhydroxide aggregates were collected in the traps. Image analysis, scanning electron microscopy and energy dispersive X-ray analysis were used to examine the aggregates.The aggregates consisted of primary particles that persisted in the aggregates. The mean diameter of the aggregates was constant with depth. The aggregates consisted predominantly of iron, phosphorus and oxygen but calcium was also an important constituent. Significant concentrations of manganese and sodium were also detected. The molar ratio Fe:P remained constant at 4:1 as did the ratio Fe:Ca at 52:1. The concentration of iron, phosphorus and calcium in the aggregates increased with depth, whilst the concentration of manganese decreased with depth in parallel with a gradient of increasing anoxia.The stable water column formed under ice cover and the temporal and spatial data provide evidence that the Fe:P and Fe:Ca ratios are constant and characteristic of the aggregates, whilst the overall composition of the aggregates is more dynamic and dependant on redox conditions and water chemistry.     

Autophagy of HSP70 and chelation of lysosomal iron in a non-redox-active form

Lysosomes contain most of the cell's supply of labile iron, which makes them sensitive to oxidative stress. To keep lysosomal labile iron at a minimum, a cellular strategy might be to autophagocytose iron binding proteins that temporarily would chelate iron in a non-redox-active form. Previously we have shown that autophagy of metallothioneins, as well as of non-Fe-saturated ferritin, meets this goal. Here we add another stress-regulated protein to the list, namely HSP70.     

Modification of ferritin during iron loading

Recombinant human ferritin loaded with iron via its own ferroxidase activity did not sediment through a sucrose-density gradient as a function of iron content. Analysis of the recombinant ferritin by native PAGE demonstrated an increase in altered migration pattern of the ferritins with increasing sedimentation, indicating an alteration of the overall charge of ferritin. Additionally, analysis of the ferritin by SDS-PAGE under nonreducing conditions demonstrated that the ferritin had formed large aggregates, which suggests disulfide bonds are involved in the aggregation. The hydroxyl radical was detected by electron spin resonance spectroscopy during iron loading into recombinant ferritin by its own ferroxidase activity. However, recombinant human ferritin loaded with iron in the presence of ceruloplasmin sedimented through a sucrose-density gradient similar to native ferritin. This ferritin was shown to sediment as a function of iron content. The addition of ceruloplasmin to the iron loading assay eliminated the detection of the DMPO-*OH adduct observed during loading using the ferroxidase activity of ferritin. The elimination of the DMPO-*OH adduct was determined to be due to the ability of ceruloplasmin to completely reduce oxygen to water during the oxidation of the ferrous iron. The implications of these data for the present models for iron uptake into ferritin are discussed.     

Lysosomal and cytosolic ferritins A biochemical and electron-spectroscopic study

Summary Cytosolic and lysosomal ferritin and haemosiderin were isolated from rat livers which had been iron-loaded by four intraperitoneal injections of iron-dextran. The cytosolic and lysosomal ferritins, prepared in a phosphate-free medium, were subjected to gel-filtration chromatography on Sepharose 613, yielding four fractions: a cytosolic monomeric (CMF) and void-volume ferritin fraction (CVVF), and a lysosomal monomeric (LMF) and void-volume ferritin fraction (LVVF). Of each fraction the following aspects were examined: (a) immunoreactivity against specific antiserum; (b) the Fe/P mass ratio and the effect of dialysis on this ratio using electron probe micro-analysis (EPMA); (c) morphology and Fespecific imaging using electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). For haemosiderin one aspect, the Fe/P ratio, was determined before and after extensive purification. The following results were obtained (a) All ferritin fractions reacted with anti- (rat liver ferritin). (b) The Fe/P ratios as determined in CMF in an haemosiderin were not affected by dialysis or extensive purification, respectively. The Fe/P ratio in CWF was affected by dialysis. In the lysosomal fractions, only a trace of phosphorus (LVVF) or no phosphorus (LMF) was detected. (c) Morphologically, CMF and CVVF were found to be rather homogeneous; the iron core diameters of both fractions were in the known size range. LMF and LVVF were of rather heterogeneous composition; the core diameters of these fractions were different. In conclusion: the phosphorus in ferritin and haemosiderin is firmly bound; Haemosiderin, when derived from ferritin, has to take up phosphorus in the lysosomes.